Chondroitinases are enzymes of bacterial origin that act on chondroitin sulfate, a component of the proteoglycans that mediate the attachment between the retina and the vitreous body of the human eye. Examples of chondroitinase enzymes are chondroitinase ABC, which is produced by the bacterium Proteus vulgaris (P. vulgaris), and chondroitinase AC, which is produced by A. aurescens. Chondroitinases ABC and AC function by degrading polysaccharide side chains in protein-polysaccharide complexes, without degrading the protein core.
Yamagata et al. (J. Biol. Chem. 243:1523-1535, 1968) describe the purification of the chondroitinase ABC from extracts of P. vulgaris. This enzyme selectively degrades the glycosaminoglycans chondroitin-4-sulfate, dermatan sulfate, and chondroitin-6-sulfate (also referred to respectively as chondroitin sulfates A, B, and C which are side chains of proteoglycans) at pH 8 at higher rates than it degrades chondroitin or hyaluronic acid. The products of the degradation are high molecular weight unsaturated oligosaccharides and an unsaturated disaccharide. However, chondroitinase ABC does not act on keratosulfate, heparin or heparitin sulfate.
Uses of chondroitinases include rapid, specific and non-surgical disruption of the attachment of the vitreous body to the neural retina of the eye, thereby facilitating removal of the vitreous body. See, for example, Hageman, U.S. Pat. No. 5,292,509.
Chondroitinase ABC is designated as chondroitinase I in the present invention. P. vulgatis chondroitinase I migrates with an apparent molecular mass of about 110 kDa when resolved by SDS-PAGE. The appearance of a doublet in SDS-PAGE resolution of chondroitinase I has been reported (Sato et al., Agric. Biol. Chem. 50:4,1057-1059, 1986). However, this doublet represents intact chondroitinase I and a 90 kDa degradation product (U.S. patent application Ser. Nos. 08/431,068; 08/428,949; 08/428,946; 08/428,948; 08/428,945; and 08/428,947, filed Apr. 24, 1995, (Atty Dock. Nos. 0646/1B017US1-0646/1B017US6) now pending). Commercial chondroitinase I protein preparations contain variable amounts of this 90 kDa degradation product and an additional 18 kDa degradation product also derived from chondroitinase I.
Another chondroitinase, chondroitinase II, has also been isolated and purified from P. vulgaris. Chondroitinase II is a polypeptide of 990 amino acids with an apparent molecular mass by SDS-PAGE of about 112 kDa. Its molecular mass as determined by electrospray and laser desorption mass spectrometry is 111,772.+-.27 and 111,725.+-.20 daltons, respectively. Chondroitinase II has an isoelectric point of 8.4-8.45. Its enzymatic activity is distinct from, but complementary to, that of chondroitinase I. Chondroitinase I endolytically cleaves proteoglycans to produce end-product disaccharides, as well as at least two other products which are thought to be tetrasaccharides. Chondroitinase II digests at least one of these tetrasaccharide products of chondroitinase I digestion of proteoglycan.
Native or wild-type P. vulgaris bacterial strains typically do not produce significant amounts of chondroitinases I and II under ordinary growth conditions. Wild-type strains of P. vulgaris can be induced to produce detectable levels of chondroitinase by providing an inducing substrate, such as chondroitin sulfate, as the sole carbon source. However, chondroitin sulfate, which is obtained from shark cartilage, is expensive and available only in limited quantities. Alternatively, cloned chondroitinase I and II genes in E. coli can be expressed using a heterologous expression system with an artificial inducer, which also increases the cost of chondroitinase I and II production.
Thus, there is a need in the art for P. vulgaris chondroitinase I and II production that does not require exogenous inducers.